ABSTRACT

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.

Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ⁹-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

摘要

ABTL0812是一种具有抗癌活性的一流小分子，目前正在晚期子宫内膜癌和鳞状非小细胞肺癌患者的2期临床试验中进行临床评估（NCT03366480）。以前，我们表明 ABTL0812 诱导 TRIB3 假激酶表达，导致 AKT-MTORC1 轴和巨自噬/自噬介导的癌细胞死亡的抑制。然而，ABTL0812 引起的细胞毒性自噬中涉及的精确分子决定因素仍不清楚。使用广泛的生化和脂质组学分析，我们证明 ABTL0812 通过削弱 DEGS1（delta 4-去饱和酶，鞘脂 1）活性来增加细胞长链二氢神经酰胺，这导致持续的内质网应激并通过 ATF4-DDIT3-TRIB3 激活未折叠蛋白反应 (UPR)，最终促进癌细胞中的细胞毒性自噬。因此，增加细胞二氢神经酰胺或与外源性二氢神经酰胺孵育的药理学操作会导致内质网应激、UPR 和自噬介导的癌细胞死亡。重要的是，我们优化了一种方法来量化来自正在进行的临床试验的患者的血液样本中的 mRNA，这些患者显示出显着增加DDIT3和TRIB3 mRNA。这是首次报道 UPR 标记物会因任何药物治疗而在人体血液中发生变化，这支持将它们用作激活人体 ER 应激的化合物的药效生物标志物。最后，我们发现 MTORC1 抑制和二氢神经酰胺积累协同诱导自噬和细胞毒性，表型复制 ABTL0812 的作用。鉴于 ABTL0812 正在临床开发中，我们的研究结果支持以下假设：操纵二氢神经酰胺水平可能代表一种针对癌症的新治疗策略。

缩写： 4-PBA：4-苯基丁酸酯；AKT：AKT 丝氨酸/苏氨酸激酶；ATG：自噬相关；ATF4：激活转录因子4；Cer：神经酰胺；DDIT3：DNA 损伤诱导转录物 3；DEGS1：delta 4-去饱和酶，鞘脂 1；dhCer：二氢神经酰胺；EIF2A：真核翻译起始因子 2 α；EIF2AK3：真核翻译起始因子2α激酶3；ER：内质网；HSPA5：热休克蛋白家族A（Hsp70）成员5；MAP1LC3B：微管相关蛋白 1 轻链 3 β；MEF：小鼠胚胎成纤维细胞；MTORC1：雷帕霉素激酶复合物 1 的机制靶点；NSCLC：非小细胞肺癌；THC：Δ ⁹ -四氢大麻酚; TRIB3：tribbles假激酶3；XBP1：X-box结合蛋白1；UPR：未折叠蛋白反应。